Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisti...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2014-07, Vol.111 (29), p.E2967-E2976
Main Authors: Port, Fillip, Chen, Hui-Min, Lee, Tzumin, Bullock, Simon L
Format: Article
Language:English
Subjects:
Alleles
Animals
Animals, Genetically Modified
Base Sequence
Biological Sciences
CRISPR-Cas Systems - genetics
DNA Repair
Drosophila melanogaster
Drosophila melanogaster - genetics
Gene Targeting
Genes, Essential
Genes, Insect - genetics
Genetic Engineering
Genetic Vectors
Genome, Insect - genetics
Genomes
Germ Cells - metabolism
Insects
Molecular Sequence Data
Mutation - genetics
Optimization
Phenotype
Plasmids
PNAS Plus
Ribonucleic acid
RNA
RNA - genetics
Transgenes - genetics
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Summary:The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line–restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25–100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1405500111